Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle that alternates between a mammal and a tick vector. Like many bacterial pathogens, B. burgdorferi must adapt to a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual segmented genome that includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded genes are critical for successful adaptation by B. burgdorferi to the different environments that the spirochete encounters during its infectious cycle. A major focus of this project is to determine how and why the Lyme disease spirochete maintains such a unique genomic structure and the specific contributions of individual plasmids and genes at each stage of the infectious cycle. As part of the in vivo adaptive response, Lyme disease spirochetes colonizing the midgut of an infected tick initiate synthesis of a plasmid-encoded, abundant outer surface protein, OspC, during tick feeding, which prepares the spirochete for transmission to a mammalian host. We have previously demonstrated that B. burgdorferi mutants lacking OspC cannot initiate mammalian infection by tick bite or needle inoculation. However, since OspC represents a potent neutralizing immune target, we and others also have shown that shortly after B. burgdorferi establishes mammalian infection, synthesis of OspC must be shut off in order to avoid clearance of the spirochete by the host's acquired immune response. In FY2011, Dr. Amit Sarkar investigated the hypothesis that a 17kb linear plasmid, lp17, encodes a repressor that negatively regulates ospC expression (3). By restoring all or part of the lp17 plasmid to a highly attenuated B. burgdorferi clone, he was able to demonstrate that a small region of lp17 is sufficient to repress ospC expression. Furthermore, by introducing a subset of lp17 sequences on a shuttle vector, Amit demonstrated that a single gene, bbd18, is responsible for this effect. Results obtained by Beth Hayes, using a recently developed LacZ reporter system (FY2010), indicate that BBD18 negatively regulates OspC at the transcriptional level, perhaps indirectly (3). The bbd18 locus on lp17 encodes an open reading frame that is well conserved among diverse B. burgdorferi strains, but with no homologs in other organisms. The general characteristics and predicted structure of BBD18 are compatible with DNA interaction, but bioinformatic analyses do not reveal homology with known binding proteins, DNA binding domains or transcriptional regulators. Ongoing studies will determine if the entire BBD18 open reading frame is needed for ospC repression in B. burgdorferi and whether BBD18 can bind DNA in a sequence-specific fashion. Other experiments will determine whether deletion of bbd18 in wild type B. burgdorferi results in continued ospC expression in vivo and abrogation of persistent B. burgdorferi infection, as predicted. Ultimately, this combined in vitro and in vivo approach should elucidate the molecular mechanisms by which the essential virulence factor OspC is regulated in the mammalian host. The complex genome of B. burgdorferi consists of a linear chromosome and more than 20 linear and circular plasmids. The 38kb linear plasmid 38 (lp38) of B. burgdorferi contains a number of genes that are differentially regulated in response to conditions mimicking the tick or mouse environments, suggesting that this plasmid may encode proteins that are important in vivo. In FY2011, Dr. Daniel Dulebohn undertook to create a B. burgdorferi clone specifically lacking lp38 while retaining the remaining plasmids known to be required for virulence, in order to investigate the contributions of lp38-encoded genes in the tick vector and mammalian host (2). Previous studies from our lab and others have demonstrated that B. burgdorferi plasmids can be selectively displaced through introduction of an incompatible shuttle vector. To this end, Dan developed a B. burgdorferi shuttle vector using the genes responsible for lp38 replication and showed that this shuttle vector effectively displaced lp38 from infectious B. burgdorferi. He found no significant differences in morphology, growth rate, protein or antigenic profile of B. burgdorferi lacking lp38 relative to wild type spirochetes. Dan also analyzed the contributions of lp38 in an experimental rodent model of infection and found that clones lacking lp38 were fully proficient to establish a disseminated infection in mice. Additionally, B. burgdorferi lacking lp38 were acquired from infected mice by feeding ticks and transmitted to naive mice at the next tick bloodmeal. These data clearly demonstrate that B. burgdorferi lacking lp38 are viable in both the tick vector and mammalian host, and readily transmitted between them (2). We find these results surprising, given the degree of conservation and regulation of lp38 genes. It is possible that within the ecology of the natural infectious cycle, which includes different host species, mixed infections and variable physical environment, conditions exist in which spirochetes carrying lp38 have a selective advantage. However, within the context of the experimental mouse-tick infectious cycle, we conclude that lp38 can be added to the growing list of conserved, highly regulated, yet expendable elements of the B. burgdorferi genome. The restriction-modification (R-M) systems of many bacteria present a barrier to the stable introduction of foreign DNA. The type strain of the Lyme disease spirochete, B. burgdorferi B31, has two plasmid-borne putative R-M loci, bbe02 and bbq67, whose presence limits transformation with shuttle vector DNA from Escherichia coli. BBE02 and BBQ67 have been classified as homologs of type II R-M enzymes, which are composed of an endonuclease-methyltransferase pair that recognizes/cleaves and modifies the same DNA sequence. Bioinformatic analyses suggest that both BBE02 and BBQ67 encode N6-adenine methyltransferases. This implies that genomic DNA should be modified in spirochetes carrying the bbe02 and bbq67 loci. In FY2011, Dr. Ryan Rego investigated the roles of the putative R-M genes in B. burgdorferi (1). He compared the transformation of B. burgdorferi with shuttle vector DNA isolated from either E. coli or B. burgdorferi, in order to determine whether the putative R-M system genes of B. burgdorferi encode activities that can discriminate between cellular and foreign DNA of identical nucleotide sequence. Ryan also examined whether and how the presence of putative R-M genes in either donor or recipient B. burgdorferi influenced transformation outcome. Finally, he analyzed the type of modification present on DNA isolated from B. burgdorferi with different R-M genes. The data from this study, published in FY2011, demonstrate for the first time that the bbe02 and bbq67 loci of B. burgdorferi encode enzymes that both methylate endogenous DNA and restrict foreign DNA lacking similar modifications in a sequence-specific fashion, as predicted for R-M systems (1). These findings have basic implications regarding horizontal gene transfer among B. burgdorferi strains with distinct plasmid contents. These results also help elucidate the molecular mechanisms underlying the relative inefficiency of genetic transformation of B. burgdorferi and suggest ways in which genetic manipulation of this pathogenic spirochete could be enhanced.